DegS initiates the envelop-stress response of gram-negative bacteria by cleaving the periplasmic domain of RseA, the major regulator of sigmaE activity. This initial event, which is activated by the binding of OMP- peptides to the PDZ domain of DegS, triggers a series of proteolysis of RseA and leads to induction of the sigmaE stress-response. In contrast to the established physiological significance of DegS and RseA in the sigmaE pathway, little is known for the molecular interactions between these two proteins. I have developed a quantitative assay to monitor the cleavage reaction in detail. I have also crystallized a DegS variant lacking the regulatory PDZ domain. The allosteric activation of DegS by OMP-peptide and the role of the PDZ domain will be first examined. The in vitro findings will be tested in vivo using genetic complementation and lacZ reporter assays. The interaction regions between DegS and RseA will be identified by crystallography and alanine scanning. I will also test whether dimeric- or trimeric RseA interacts better with DegS. Finally, I will investigate how DegS influences the next cleavage of RseA by RseP. This research will enhance our understanding of proteolytic signal transduction pathways and potentially open doors for novel anti-microbial and anti-cancer treatments. DegS is essential for the survival of E. Coli, and is an indispensible component of the sigmaE stress response. Notably, the sigmaE pathway is required for the virulence of many pathogenic bacteria. Thus, understanding how DegS recognizes activators and substrates could open doors for developing novel anti-microbial agents. Moreover, since the mammalian ortholog of DegS, HtraA2/Omi, is associated with apoptosis and oncogenesis, my studies may have implications for cancer.